Affinity purification ("affinity chromatography") broadly refers to separation methods based on the relatively high binding capacity ("affinity") of a target material to be purified, generally termed a "ligate," for a complementary ligand. Affinity separation is a preferred method for purifying proteins and other biomolecules from complex, biologically derived fluids. The key to the method's attractiveness is its unequaled degree of selectivity.
Affinity separations, as they are conventionally practiced, typically involve a number of sequential steps. First, a solution containing a component to be separated from the solution (a component of interest) is passed through a column containing a highly specific ligand immobilized on a support, usually a membrane or high-surface-area beads or particles. As the fluid passes through the column in this loading step, the desired component binds selectively and reversibly to the immobilized ligand, while most impurities pass unhindered. Residual impurities are removed by flushing the column with an appropriate buffer solution in a subsequent washing step. The component, now purified but still bound to the immobilized ligand, is then recovered by passing an eluent solution through the column that has the effect of disrupting the ligand-to-ligate binding interaction. Generally, the pH, concentration of a salt, or some other chemical characteristic of this eluent solution is altered significantly from the corresponding values of the loading and wash solutions, and it is this change that is responsible for weakening the affinity interaction and thereby causing desorption and elution of the ligate molecule.
Numerous products and procedures have been developed for the preparation of solid membrane and particle-based affinity supports to retain immobilized protein ligands for separations. See, "Affinity Chromatography: Principles and Methods," Pharmacia LKB Biotechnology, 1988; Scouten, W. H. "Affinity Chromatography: Biospecific Adsorption on Inert Matrices," Wiley Interscience, 1981; and "Methods in Enzymology," Vol- XXXIV, Affinity Techniques, Jacoby, W. B. and Wilchek, M., Eds., Academic Press, 1974. Important parameters for these products and procedures include: (i) the maximum amount of ligand that can be immobilized on the affinity support, (ii) the percent yield with which the ligand can be immobilized, and (iii) the degree to which the functionality of the ligand (i.e., the ability of the ligand to bind target proteins to the ligand) is preserved once the ligand has been immobilized. All of these influence the binding capacity of the affinity support and the cost of preparing it.
The literature also describes various approaches to increasing the ligand binding capacity and efficiency while preserving ligand biological function. See for example, D. Weber et al, J. Chromatography, 510, 59-69 (1990). These approaches include: (i) varying the amount and type of the functional groups present on the support, (ii) optimizing the conditions of pH and ionic strength for attaching the ligand molecule to the support, and (iii) so called "site-directed" methods which aim to bind the ligand molecule to the support at a site on the ligand that is not involved in interaction with the target protein molecule. All of these approaches, however, are subject, among others, to the following limitations:
1. The maximum amount of ligand protein that can be bound onto a porous support is a function of the surface area on the support available for the functional groups, with higher surface area support matrices generally having more functional groups and normally binding more ligand. High surface area is usually achieved by decreasing the pore size of the support matrix. If the pore size is decreased beyond an optimum value, however, exclusion of either the ligand or the target protein will occur and the capacity of the matrix will be adversely affected. See, Narayanan, S. R., et al., J. Chromatography, 503, 93-102 (1990).
2. As the amount of ligand presented during immobilization to the support matrix approaches the maximum ligand binding capacity of the support, the efficiency of immobilization begins to decrease. Therefore, it becomes costlier in terms of wasted ligand to produce supports that contain approximately the maximum amount of ligand that can be bound. See, e.g., Nakamura, K., et al., J. Chromatography, 510, 101-113 (1990).
3. As the density of ligand protein on the support is increased, the target protein binding capacity per gram of immobilized ligand begins to decrease, presumably due to steric factors that reduce the accessibility of the target protein molecules to the binding sites on the immobilized ligand. See, e.g., Comoglio, S., et al., Biochem. Biophys. Acta, 420, 246-257 (1976) and Tharakan, J. P., et al., J. Chromatography, 522, 153-162 (1990).
A need therefore exists for methods to immobilize protein ligands, providing an increase in the maximum amount of ligand that can be immobilized on the support without appreciable loss of biological function of the ligand, i.e., without loss of its target protein binding capacity. This would enable production of higher capacity affinity supports as well as more efficient and less costly production of affinity supports.